In all eukaryotes, numerous pathways and signaling networks depend upon the protein ubiquitin (Ub) to regulate the amounts, localization, interactions, or activities of thousands of proteins. Ub is the archetype of a family of proteins tat regulate other proteins by covalent attachment. Like other post-translational modifications, Ub conjugation is reversible and tightly controlled. Ub and polyUb signals are used in diverse processes that include intracellular protein degradation, cell cycle control, inflammation and innate immunity, protein trafficking, chromatin structure and gene expression, and DNA damage response pathways. Considerable progress has been made to define the structural and biochemical bases for Ub's role in these and many other processes, yet our picture of how Ub-mediated pathways are controlled and interact is woefully incomplete. The fundamental premise of this proposal is that knowing the levels of free and conjugated ubiquitin under conditions of cell growth or stress, and how the flux of Ub through those pools is regulated, are necessary if we are to understand intracellular Ub-dependent signaling. Our Aims combine method and resource development with focused investigations of Ub homeostasis, polyUb stability and editing, and the regulation of histone ubiquitination. A specific long-term goal is to test the ide that changes in Ub flux through specific protein conjugates can identify regulatory nodes of signaling pathways, even in cases where steady-state levels of those conjugates do not change. We propose to develop and apply new tools that, for the first time, will enable quantitative measurements of (1) the distribution of free or conjugated Ub in live cells, and (2) the movement of Ub through specific protein conjugates. These measurements will be made in cultured yeast or mammalian cells to evaluate Ub dynamics during growth and in response to various stress conditions, to monitor the absolute flux of Ub through different forms of polyUb and Ub-histone conjugates, and to determine globally the relative fluxes (i.e., stabilities) of Ub among the cell' inventory of Ub-protein conjugates. A mathematical model developed to describe Ub's movement through its different biochemical forms will be refined based on measurements of intracellular Ub concentrations and flux. The ability of the model to predict effects of mutations and other perturbations of Ub pathways will be tested to evaluate its accuracy and utility.